Both mechanical ventilation and hyperoxia, although necessary supportive interventions, have been independently implicated in the genesis of lung injury. Although patients with hypoxemic respiratory failure are routinely exposed to both of these interventions, little is known about their potential adverse interaction. We hypothesize that cyclic stretch of alveolar epithelial cells caused by mechanical ventilation, in the presence of moderate hyperoxia, causes an increase in production of reactive oxygen species (ROS) that is greater than that produced by either stimulus alone. These excess ROS, through altered oxidative signaling, result in increased apoptotic cell death of alveolar epithelial cells. The resulting cell loss leads to the breakdown of alveolar-epithelial barrier integrity causing the lungs to become more permeable to fluid, cells, and inflammatory mediators. This influx of fluid and cells causes adverse derangement in lung mechanics and gas exchange, and the accelerated development of lung injury. In the proposed studies, we will use an in vivo model of lung injury caused by the combined effect of large tidal volume mechanical ventilation and moderate hyperoxia, an in vitro system that allows cyclic mechanical stretch of isolated alveolar epithelial cell monolayers in hyperoxic conditions as well as isolation of alveolar type II epithelial cells from rats after exposure to mechanical ventilation and hyperoxia. We will use these systems to accomplish the following specific aims: Specific Aim 1: To demonstrate that the combination of mechanical stretch and moderate hyperoxia leads to early loss of alveolar-capillary barrier integrity and accelerated development of lung injury. Specific Aim 2: To determine the mechanisms by which ROS are produced during mechanical stretch and hyperoxia in cultured alveolar epithelial cells. Specific Aim 3: To demonstrate that COMBINED mechanical stretch and moderate hyperoxia increases alveolar type II epithelial cell apoptosis via activation of apoptosis signal-regulating kinase-1 (ASK-1) and to determine the effect of ASK-1 mediated apoptosis on alveolar epithelial barrier integrity. Because ALI patients supported with mechanical ventilation receive varied levels of supplemental oxygen, the proposed work could have significant clinical applicability, including the identification of potential therapeutic targets for the prevention and/or treatment of acute lung injury.